Weighted collagen microsponge for immobilizing bioactive material

ABSTRACT

Weighted collagen microsponges having a highly crosslinked collagen matrix are described suitable for use in culturing organisms in motive reactor systems. The microsponges have an open to the surface pore structure, and pore sizes and volumes suitable for immobilizing a variety of bioactive materials. The microsponges also have an average particle size in the range of about 100 to about 1000 microns and a specific gravity above about 1.05.

This invention was made in the course of, or under, a contract with NIH.The government has rights to the invention pursuant to SBIR Grant No.CA37430.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention pertains to the art of immobilizing bioactivematerials and particularly relates to a collagen microsponge for use inmotive bioreactor systems. The present invention also relates to the artof culturing microorganisms and cells, hereinafter referred tocollectively as organisms, and particularly relates to the culturing oforganisms immobilized on microsponges in motive reactor systems assubmerged suspensions.

2. Description of the Prior Art

Various arrangements for immobilizing bioactive materials are known.Solid supports have long been used for immobilizing microorganisms inthe treatment of waste water and related fermentation processes. Morerecently, solid mircocarriers have been used to obtain high celldensities in the culture of attachment-dependent cells. For example,microporous polymeric supports fabricated for example from dextran havebeen used for cultivating cells. Such supports can be obtainedcommercially from Pharmacia Fine Chemicals under the brand nameCytodex.sup.®. Such solid bio-supports, however, are not suitable formotive reactor systems such as vigorously stirred tanks and fluidizedbeds since substantially all of the cells are adherent to the surface ofsaid supports and thus are exposed to impact stress and trauma duringoperation.

Porous inorganic microcarriers also are known and such supportspotentially provide protection for the cells in motive applicationssince the cells populate the interior of the microcarriers.Unfortunately, inorganic microcarriers cannot be made with the propercombination of permeability and specific gravity to function well in allmotive applications. For example, the porous fritted glass or cordieritesupports described in Messing et al. U.S. 4,153,510 would typicallyexhibit specific gravities in aqueous suspension of less than about 1.3if their void fractions are greater than about 80% (Note that voidfractions for the Messing supports are not disclosed). Quiteunderstandably, these supports are not suitable for all motive reactorsystems where a higher specific gravity generally is needed to ensurehigh relative velocities for maximum rates of mass and energy transfer.Consequenty, these supports have generally been relegated for use inpacked bed applications.

An object of the present invention is to provide a microspongecontaining immobilized bioactive materials suitable for use in motivereactor systems.

Another object of the present invention is to provide a microspongesuitable for immobilizing a large variety of organisms characterized bywide variations in size and their degree of attachment to solidsupports.

A further object of the present invention to provide a microspongesuitable for motive reactor systems which is conducive to maximizing themetabolic activity of immobilized organisms.

Yet another object of the present invention is to provide a method forcontinuously culturing organisms at high concentrations.

Still another object of the present invention is to provide amicrosponge suitable for motive reactor systems which permits theculturing of organisms at high concentrations while accommodating eithermaximum growth rate or maximum metabolic activity.

These and other objects of this invention will become apparent from aconsideration of the specification and appended claims.

SUMMARY OF THE INVENTION

The present invention pertains to a weighted collagen microsponge forimmobilizing bioactive materials in motive bioreactor systems, saidmicrosponge comprising a porous, biostable, highly corsslinked collagenmatrix containing an inert weighting material, said collagen matrixhaving an open to the surface pore structure with an average pore sizein the range of from about 1 micron to about 150 microns, with the poresof said matrix occupying from about 70 to about 98% by volume of themicrosponge, said microsponge also having an average particle size offrom about 100 microns to about 1000 microns and a specific gravity ofabove about 1.05.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph showing a suitable collagen microspongematrix of the present invention illustrating a fibrous structure.

FIG. 2 is a photomicrograph of another collagen microsponge matrixaccording to the present invention illustrating a leafy structure.

DETAILED DESCRIPTION

The present invention is directed to a weighted collagen microspongecontaining immobilized bioactive materials, particularly organisms,suitable for use in motive bioreactor systems. As used throughout thespecification and claims, the term "bioactive material " broadlyencompasses both enzymes and other chemical factors such as chelatingagents, hormones, antibodies, etc., and organisms, i.e., microorganismsand the cells of higher organisms. The organisms may be either living ordead and may be derived without limitation from such diverse sources asbacteria, fungi, viruses, algae, yeasts, animal cells (tissue), e.g.,mammals, insects, fish and plant cells. Since the invention hasparticular advantages when used for culturing organisms, it generallywill be described with reference to such embodiments, although it is notto be so-limited.

The microsponge of this invention is formed of a highly crosslinkedcollagen. The modified (crosslinked) collagen of this invention isbiocompatible (non-toxic) and is stable in service for an appropriateperiod of time, e.g., on the order of months. Biocompatibility refers tothe ability of the collagen matrix material to support a viable cultureof organisms without substantially adversely affecting any desiredcharacteristic of the immobilized organisms, e.g., in the case ofhybridomas, the collagen matrix material must not reduce the productionof momoclonal antibodies. The stability or biostability of the matrixmaterial refers to tis ability to maintain its strength and integrityunder in vitro conditions over the relevant time period for culturingthe organism of interest. For example, in the case of a hybridomaculture for producing monoclonal antibodies, it is expected that themotive bioreactor would be operated continuously for three to six monthsor more. Thus, the matrix material should be biostable for this timeperiod.

Collagen is a biodegradable polymer found in animals, including man. Ithas numerous uses in the medical art and in most applications isreconstituted and crosslinked into an insoluble form using variouscrosslinking agents, often glutaraldehyde. Unfortunately, many if notall of the commonly used crosslinking agents, particularlyglutaraldehyde, which are unavoidably present in the crosslinkedcollagen, cause adverse biological effects and so are cytotoxic.

Recently, a new collagen-based matrix of improved biostability has beendiscovered. This collagen material is prepared without the conventionalcrosslinking agents. In this matrix, Type I, II or III collagen iscrosslinked using a carbodiimide or a succinimidyl active ester and/orsevere dehydration conditions at temperatures ranging from 50° to 200°C. Such crosslinked collagen typically has a molecular weight of fromabout 1×10⁶ to in excess of 50×10⁶. The molecular weight of the collagenbetween adjoining crosslinks varies from about 1000 to 100,000 via theformation of covalent bonds. Because of its resistance to degradation bycollagenase and other proteinases, this crosslinked collagen has beenfound to be particularly suitable as the porous matrix of themicrosponge. In fact, when used for the continuous culture ofimmobilized organisms, particularly hybridoma cells expressingmonoclonal antibodies, microsponges made from this collagen materialhave exhibited some surprising properties. For example, hybridomascultured with essentially protein-free medium are much more effective inproducing monoclonal antibodies when immobilized on and/or in suchcollagen matrix microsponges than when such matrix is not present.Furthermore, microsponges made from the noted crosslinked collagenmaterial appear to preferentially retain high concentrations of living(viable) cells and expel non-viable cells.

The preferred crosslinked collagen can be prepared from both solublecollagens and insoluble collagens of the Types I, II and III. Thesoluble collagens are prepared by limited enzymatic digestion and/orextraction of tissue enriched in such collagen types. Insolublecollagens are derived from the following typical sources: Type Icollagen: bovine, porcine, chicken and fish skin, bovine and chickentendon and bovine and chicken bones including fetal tissues; Type IIcollagen: bovine anticular cartilage, nasal septum, sternal cartilage;and Type III collagen: bovine and human aorta and skin. For example,Type I collagen from bovine corium may be used. It is preferred to useType I tendon collagen.

In order to be suitable for culturing high concentrations of organismsin motive reactor systems and allow for the transfer of nutrients to theimmobilized organisms and the transfer of desired products from themicrosponge, the collagen microsponge of the present invention mustsatisfy several functional requirements. The microsponge typically is inthe shape of a bead and should have a particle size within the range ofabout 100 microns to 1000 microns, preferably from about 200 microns to500 microns. At larger particle sizes the entire internal volume of theporous structure is not utilized effectively for producing the desiredproduct by reaction between the immobilized bioactive material and theliquid medium contacted therewith, thus degrading the volumetricproductivity of the motive reactor employing such microsponges. Smallerparticle sizes present practical problems in preparing the microspongeand in operating the motive reactor.

Permeability of the microsponge is another important consideration. Amicrosponge's permeability is determined by the interrelationship of itsporosity or void fraction and its pore structure. Void fraction isdefined as the ratio of the volume of interstices of a material to thetotal volume occupied by the material and often is expressed as apercentage. In order to permit operation at high organismconcentrations, the microsponge should have a void fraction of betweenabout 70% and 98%. Preferably the void fraction of the collagenmicrosponge is greater than 85% and most desirably is greater than about90%.

The microsponge also must possess an open to the surface port structure.This allows for cell entry, without excessive shear forces, cellretention, subsequent cell growth, and expulsion of excess cell mass.For example in cases where the desired product is not secreted by theorganisms, e.g., genetically engineered E. coli with a nonexpressed rDNAproduct such as insulin, the organisms must be able to escape themicrosponge as the immobilized colony expands by division. An open porestructure is essential if this process is to proceed on a continuousbasis, without rupturing the microsponge structure. The desired organismproduct is recovered as an entrained component of the culture harvestliquor.

The microsponge should contain pores with an average size within therange of about 1 micron for the smallest microbes and for viruses, up toabout 150 microns for large mammalian and plant cells. Generally, thepores of the microsponge must be at least as large as the smallest majordimension of the immobilized bioactive material but less than about 5times the largest major dimension. Preferably, the pore size of thematrix is on the order of 1.5 to 3 times the average diameter of theorganism or cell. If unknown, the smallest and largest major dimensionsof an organism can be determined using known techniques. Applicants havefound that the recited combination of particle sizes and port sizesinsure adequate mass transfer of constituents such as nutrients to theimmobilized organisms, as well as adequate mass transfer ofconstituents, such as desired metabolites from the immobilizedorganisms.

For use in motive reactor systems, the collagen microsponge also must beweighted. The crosslinked collagen used as the matrix material in thepresent invention generally has a specific gravity of about 1.0 of less.For proper operation in a motive reactor, a specific gravity of aboveabout 1.05, preferably above about 1.3 and most preferably between about1.6 and 2.0 is desired. It has been found surprisingly that it ispossible to obtain collagen microsponges of the proper specific gravityby introducing certain weighting additives into the microsponge withoutundesirably reducing its void fraction. The weighting additive must besubstantially inert in the reactor environment and non-toxic to theimmobilized organism, or must be suitably treated to render the additivenon-toxic. Also, the weighting additive should not adversely affect theproductivity of the immobilized organism. Generally, materials, such asmetals and their alloys and oxides, and ceramics, having a specificgravity above about 4 and preferably above about 7 are used. Examples ofsuitable weighting additives for use in the broad practice of thisinvention are chromium, tungsten, molybdenum, cobalt, mickel, titaniumand their alloys, e.g., Monel, 316 stainless, Vitalium (a cobalt alloywith chromium and molybdenum), titanium 6Al-4V (a titanium alloy withaluminum and vanadium) and Haynes Stellite Alloy 25 (a cobalt alloy withchromium nickel, tungsten and manganese). Many of these materials,however, may not be compatible with certain organisms and routineexperimentation will be necessary to assess toxicity for anyapplication. For example, titanium is the weighting material of choicewith hybridomas, since most other metals are cytotoxic.

The weighting additive can be introduced into and dispersed throughoutthe microsponge as a finely divided powder, with most particles having asize on the order of 10 to 40 microns. However, to minimize the surfacearea of the weighting additive, it is desirable to employ it as a solidcore in the microsponge. Sufficient weighting material is added to yielda microsponge with the desired specific gravity. For example, about a 50micron diameter core of a weighting additive having a specific gravityof about 7.0 coated with a 50 micron thick layer of collagen having anaverage pore size of 20 to 40 microns and a void fraction of about 99%yields a microsponge with a specific gravity of about 1.7 having anoverall void fraction of about 85%. Such a microsponge is particularlysuitable for use in an aerobic motive reactor systems.

Finally, for motive applications the collagen microsponge should exhibitthe proper resistance to attrition. A charge of microsponges preferablyshould have a useful life on the order of three to six months or more.Typically, the microsponges should exhibit not greater than about a 10%loss in volume after three months of operation.

Normally, organisms exhibit wide variation in their degree of attachmentto solid supports. Certain organisms, for example, readily cling orattach to a wide variety of supports, including both organic andinorganic materials, while others will only attach to supports ofbiological origin (attachment-dependent organisms). Other organismsexhibit little direct attachment to any support material(attachment-independent organisms). The collagen microsponge of thepresent invention, because it is prepared from a natural polymer andbecause of its permeability (porosity and pore structure) should besuitable for immobilizing substantially all types of organisms.

In fact, as noted in more detail below, it even is possible to tailorthe micro-structure or configuration of the microsponge to bestaccommodate the attachment tendency of the immobilized organism. Forexample, microsponges having the wire-mesh structure (FIG. 1) can beemployed in conjunction with attachment-independent organisms, whilemicrosponges exhibiting a leafy structure (FIG. 2) can be used withattachment dependent organisms.

Any suitable procedure used by the prior art for immobilizing suchorganisms on microsponges can be used in the present invention includingsuch techniques as adsorption and chemical coupling. For example, in thecase of certain organisms it will only be necessary to mix the collagenmicrosponges in a broth inoculated with the specific organism. After ashort period of time, the organism will colonize the microsponges andbecome entrapped in their pores. In the case of some organisms such asfibroblasts and hybriodomas, it also may be desirable to coat themicrosponge with attachment-promoting materials such as fibronectin,polylysine and anti-hybriodoma antibodies prior to inoculation. Othertechniques, such as applying a net charge to the surface of themicrosponge, also can be used to enhance immobilization.

As will be recognized by those skilled in this art, in the broadpractice of the present invention, the procedure used for bringing theimmobilized bioactive material into direct contact with a liquid reagentstream such as a growth supporting medium for culturing of immobilizedorganisms is not critical and any of the numerous arrangements availablein the prior art including such well known apparatus as stirred tankreactors, fixed bed reactors, fluidized bed reactors and moving bedreactors and the like could be used. Generally, when culturing organismsthe microsponges are charges to a suitable reactor and mixed thereinwith a nutrient broth and an inoculum of the organism. The microspongesshould be completely submerged. The microsponges are incubated so thatthe organisms grow and colonize the porous matrix of the microsponge.Fresh nutrient broth along with other materials necessary for growth,such as oxygen in the case of aerobic organisms, are supplied in acontinuous manner to the reactor and harvest liquor containing thebiochemical product of interest is recovered. The biochemical productmay comprise a primary or secondary metabolite of an immobilizedorganism, excess biomass generated by an immobilized organism containingfor example a non-secretory product, an immobilized enzyme catalyzedreaction product or the like.

A particular advantage of the microsponge of the present invention isthat it can be used in a mixed or motive system such as a fluidized bedreactor. As used herein, the term "motive reactor" refers to reactorsystems in which relative motion between the microsponge and the fluidmedium is provided in part by imparting motion to the microspongesthemselves. Such reactor systems substantially enhance mass and energytransfer. A particularly preferred motive reactor system is described inco-pending U.S. Pat. Application Ser. No. 706,872 filed on Feb. 28, 1985in the names of Robert C. Dean, Jr., Peter V. Grela and Subhash B.Karkare.

To prepare the highly crosslinked collagen microsponge, a suitablecollagen source first is milled to a small "fiber" size. Generally, thecollagen is milled (e.g., using a Wiley mill) to obtain particles(fibers) with a maximum dimension on the order of about 200 microns.Preferably, the collagen source material is milled (i.e., dry ground) toyield fibers having a diameter on the order of 1 to 50 microns and alength no greater than about 200 microns. Proper milling of the collagensource material is important for obtaining microsponges of the desiredstructure.

The milled collagen then is formed into a collagen-based solution ordispersion, i.e., a soluble collagen dissolved in a solvent, or aninsoluble collagen dispersed in a solvent by admixture with a suitablesolvent, particularly acids such as dilute hydrochloris acid, diluteacetic acid or the like. In the present invention organic acids areparticularly preferred, including acetic acid, lactic acid, proprionicacid, butyric acid and the like. Certain long chain fatty acids alsocould be used. The collagen is mixed into the liquid (solvent) usingstandard mixing equipment. Preferably, in the case of a collagendispersion the mixing is accomplished with a high level of agitationusing, for example, a Waring blender, so as to produce microfibers ofthe collagen. The mixture of collagen and solvent typically comprisesbetween about 0.5% to about 1.5% by weight of the collagen. The mixturepreferably exhibits a pH in the range of about 2.0 to about 4.0. A pH inthe range of 1.0 to 2.0 may also be used as long as the temperature ofthe mixture is sufficiently reduced (e.g., about 4° C.) to avoiddenaturization of the collagen.

Next, the weighting additive is blended with the collagen-liquid mixtureand the composite mixture is formed into small droplets and rapidlysolidified by freezing at a temperature below about 0° C. and preferablybelow about -30° C. to form particles of the desired size. Any knowntechnique for producing small particles can be employed in carrying outthe present invention. Suitable techniques include, inter alia, pressureor air-shear spraying emulsification techniques, droplet formation usingRaleigh liquid jet instability techniques, extrusion techniques, dropletformation using gravity or centrifugal forces, electrostatic dropletformation, and droplet formation using inertial forces. For example,suitably sized particles have been prepared using inertial forces toform small droplets at the orifice of a vibrating needle. The dropletscan be frozen by allowing them to fall into a cryogenic bath of liquidnitrogen. Obviously other chilling baths for freezing the droplets couldbe used, e.g., chilled ethanol. Also, larger sized particles formed byfreezing possibly could be reduced to the desired particle size by suchdestructive techniques as grinding and the like. Still additionaltechniques such as various coating methodologies, could be used to formmicrosponges having a solid core of the weighting additive. In this casea shell of the collagen matrix would surround the weighted core. Thoseskilled in the art will recognize other techniques suitable for formingsmall particles of the types described above and the present inventionis not intended to be limited to any specific technique.

The pore size and structure of the collagen microsponge is influenced bya variety of factors. For example, changes in the collagen concentrationappear to affect pore size, with higher collagen concentrations tendingto yield smaller pore dimensions. The pH of the mixture and the specificacid used in preparing the mixture also affect the pore size andstructure of the resultant microsponge. For example, it has been foundthat too low a pH tends to significantly limit the pore dimensions ofthe microsponge while higher pHs cause a distinct collagen phase toseparate from the original solution or dispersion thereby preventing theformation of a porous structure and when a finely divided weightingadditive is used it tends to remain in the dispersed phase. The rate offreezing also appears to influence the structure of the microsponge andthe structure also will vary with changes in collagen type.

Thereafter, the frozen composite is vacuum freeze-dried preferably usingconventional equipment operating at a vacuum of more than about 50millitorr and at a temperature in the range of about 22° C. to -100° C.The combination of freezing and drying is referred to as lyophilization.

The freeze-dried collagen matrix composite then is treated so as tocrosslink the collagen. The collagen can be crosslinked using eitherchemical crosslinking agents preferably selected from the groupconsisting of a carbodiiamide or N-hydroxy succinimide-derived activeesters (succinimidyl active esters), by severe dehydration at anelevated temperature or by a combination of these treatments. Thestrength and biostability of the collagen matrix so-prepared isinfluenced by the degree of crosslinking introduced through suchtreatment. These crosslinking methods provide a collagen matrix that issurprisingly resistant to collagenase and other enzymatic degradationthereby making these materials particularly suitable for culturingorganisms. Examples of carbodiiamides which can be used in the chemicaltreatment are cynamide and 1-ethylb l-(3-dimethylaminopropyl)-carbodiiamide hydrochloride. Suitablebifunctional succinimidyl active esters include bifunctional N-hydroxysuccinimide, 3,3'-dithio(sulfosuccinimidyl) proprionate andbis(sulfosuccinimidyl) suberate. When using such chemical crosslinkingagents, the dry collagen matrix material is immersed in a solution ofthe crosslinking agent at about room temperature for a period of time offrom about 2 to 96 hours. The solution of crosslinking agent may containfrom about 0.1 to about 15% (weight per volume) of the crosslinkingagent. Alternatively, the crosslinking agent could be added to theoriginal solution or dispersion of the collagen source.

To crosslink the collagen matrix using severe dehydration, themicrosponge is subjected to a vacuum of about 50 millitorr or more for aperiod of time of from about 2 to about 96 hours at a temperature in therange of about 50° C. to about 200° c., e.g., about 100° to 110° c.

As noted above, the two treatments also can be used in combination andit is preferred to use a severe dehydration treatment followed bychemical treatment to crosslink the collagen matrix. Generally, however,in those cases where chemical treatment precedes the dehydrationtreatment the crosslinking agent should be added directly to theoriginal collagen solution or dispersion prior to formulation of thematrix particles and lyophilization in order to facilitate subsequentvacuum dehydration treatment. Also as noted above the strength andbiostability of the collagen matrix is influenced by the degree ofcrosslinking introduced through such treatment. These crosslinkingmethods provide a collagen matrix that is surprisingly resistant tocollagenase and other enzymatic degradation thereby making thesematerials particularly suitable for culturing organisms. Wheneverchemical treatment is used, the collagen matrix should be washedextensively prior to further use in order to remove any excesscrosslinking agent. Further information concerning the procedure forpreparing the highly corsslinked collagen can be obtained from U.S. Pat.No. 4,703,108 issued Oct. 27, 1987, based on U.S. Pat. Application Ser.No. 843,828 filed on Mar. 26, 1986, a continuation of U.S. Pat.Application Ser. No. 593,733 filed on Mar. 17, 1984, now abandoned, inthe names of Frederick H. Silver, Richard A. Berg, David E. Birk, KevinWeadock and Conrad Whyne and entitled "Biodegradable Matrix and Methodsfor Producing Same," the disclosure of which is incorporated herein byreference.

After thoroughly washing the crosslinked collagen matrix in ultra-purewater, the microsponges then may be sterilized using conventionalsterilization techniques. A particular advantage of the collagenmicrosponges is that they are manufactured separate from the step oforganism immobilization and as a result they can be properly sterilizedprior to being inoculated or stored. Preferably, the microsponges aresterilized using gamma irradiation. Ethylene oxide also may be used asan alternative, as may additional sterilization procedures known tothose skilled in the art, as long as the important characteristics ofthe microsponge are not compromised. Obviously, when sterilizing themicrosponges using ethylene oxide the particles must be thoroughlyventilated in order to remove all traces of this sterilizing agentbefore subsequently using the microsponges for culturing organisms. Italso has been discovered that the severe dehydration treatment for anextended time at an elevated temperature used to crosslink the collagenmay satisfactorily sterilize the microsponge, thus obviating anyadditional treatment.

Preferably, the sterilized microsponges are aseptically packaged fordelivery to the ultimate consumer. The user simply places themicrosponges into a previously sterilized reactor, adds the propernutrients and inoculum and initiates operation. In a preferredembodiment, the package actually comprises a disposable reactor vesselhaving the necessary connections for feeding a nutrient stream, forremoving a harvest liquor and for ancillary operations, as needed, suchas heat exchange, oxygenation and process control. For a fluidized bedreaction, the vessel also would contain a suitable designed distributionplate. Such a pre-packagaed disposable reactor vessel may have a volumebetween about 0.1 liter and 10 liters. In this case, the user of thereactor simply integrates it into the process equipment consisting ofpumps, valves, piping, heat and gas exhangers and variousinstrumentation and related probes and begins operation. Providing adisposable reactor, pre-packaged with the microsponges sterilized andready for use, significantly simplifies start-up procedures forculturing organisms, particularly when changing from one culture toanother.

Although not completely understood, it has been observed that variationsin process parameters lead to important variations in the structure orconfiguration of the highly crosslinked collagen matrix itself. FIGS. 1and 2, which are photomicrographs of the collagen matrix prepared usingthe techniques described above, illustrate these different structures.The photomicrographs of FIGS. 1 and 2 were obtained using scanningelectron microscopy. FIG. 1 illustrates a collagen matrix havingsubstantially a wire-mesh structure. In this structure, the diameter ofthe fiber network typically is on the order of about 1 micron. Thisstructure is particularly desirable for attachment-independent typeorganisms such as hybriodomas. In this matrix, such organisms becometrapped in and on the matrix structure. FIG. 2 illustrates a leaf-typematrix structure. The leaves of this structure typically have athickness on the order of about 1 micron. This structure is particularlysuitable for attachment-dependent cells such as fibroblast cells.Currently, it is believed that the rate of chilling (freezing) thedroplets in the bead making process influences the morphology of thecollagen microsponge.

The following examples are intended to more fully illustrate theinvention without acting as a limitation on its scope.

EXAMPLE 1

This example describes the preparation of microsponges of the highlycrosslinked collagen matrix material.

Partially purified tendon collagen was milled to obtain fibers having alength of less than about 200 microns and a diameter of between about 5and 50 microns using a Wiley Mill obtained from VWR Scientific. Anamount of milled collagen material then was mixed with a solution ofacetic acid using a Waring blender so as to produce a collagen-baseddispersion having a pH of about 3.0 and about 1.0% (by weight) ofcollagen. Then, an inert weighting material, titanium, was added to thecollagen-based dispersion as a fine powder having particle sizes withinthe range of about 5 to about 180 microns.

The composite mixture then was formed into solid particles by firstproducing small droplets of the composite mixture. Droplets wereproduced by flowing the composite mixture through a vibrating hollowneedle having an internal diameter of about 1.3 millimeters vibrated ata frequency of about 90 Hz. The droplets fell into a cryogenic bath ofliquid nitrogen and were rapidly frozen. The frozen droplets of thecomposite mixture then were vacuum dried using a Virtis FreezemobileLyophilizer Model 6 at a vacuum of about 10 millitorr for about 48hours.

After such lyophilization, the dried microsponges were subjected to asevere dehydration (dehydrothermal treatment) at a temperature of about100° C. under a vacuum of about 10 millitorr for about 72 hours using aVWR Scientific drying oven. The microsponges then were treated with a1.0% (by weight) solution of cyanamide as a chemical crosslinking agentat a pH of about 5.5 for about 24 hours at about 20° C.

The highly crosslinked microsponges then were thoroughly washed forabout 24 using ultra-pure water, were dried and then sterilized by gammairradiation. The microsponges had particle sizes within the range ofabout 200 to 800 microns, a void fraction on the order of about 77%,pore sizes on the order of about 20 microns, and a specific gravity onthe order of about 1.1. The microsponges had a wire meshmicro-structure.

EXAMPLE 2

The microsponges of Example 1 can be used to support the growth ofhybridoma cells. In particular, about 300 ml of the microsponges can becontained in a 600 ml reactor vessel. The microsponges can be inoculatedwith the hybriodoma cells and cultured using a suitable nutrient medium.The reactor can be operated at a solids concentration of about 25≧40%,while the content of the reactor is vigorously agitated. A nutrientmedium such as Delbecko Modified Eagle medium with 10% fetal calf serumcan be passed into the reactor in a continuous manner and a productstream containing the monoclonal antibodies can be recovered at asubstantially equivalent flow rate.

It will be obvious to one of ordinary skill that numerous modificationsmay be made without departing from the true spirit and scope of theinvention which is to be limited only by the appended claims.

We claim:
 1. A weighted collagen microsponge for immobilizing bioactive materials in motive bioreactor systems, said microsponge comprising a porous, biostable, insoluble highly crosslinked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pores structure with an average pore size in the range of from about 1 micron to about 150 microns, the pores of said matrix occupying from about 70 to about 98% by volume of the microsponge, said microsponge also having an average particle size of from about 100 to about 1000 microns and a specific gravity above about 1.05, wherein said highly crosslinked collagen matrix is prepared by the steps of(a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) freezing droplets of the weighted acid/collagen mixture to form a solid matrix, (e) vacuum drying said solid matrix to form a dry matrix; and (f) crosslinking the collagen in said dry matrix by a treatment selected from the group consisting of (i) contacting said collagen with a crosslinking agent selected from the group consisting of carbodiimides and bifunctional succinimidyl active esters, (ii) subjecting said collagen to elevated temperatures under a vacuum, and (iii) a combination thereof.
 2. The microsponge of claim 1 having immobilized therein bioactive material selected from the group consisting of enzymes, dead cells and living cells.
 3. The microsponge of claim 2 wherein the cells are microorganisms.
 4. The microsponge of claim 1 wherein said collagen is Type 1, tendon collagen.
 5. The microsponge of claim 1 wherein said inert weighting material is selected from the group consisting of metals, metal alloys, metal oxides and ceramics.
 6. The microsponge of claim 5 wherein said weighting material has a specific gravity of above about 4.0 and said microsponge has a specific gravity of above about 1.3.
 7. The microsponge of claim 6 wherein said inert weighting material is dispersed throughout said collagen matrix as finely divided powder.
 8. The microsponge of claim 6 wherein said weighting material is centrally disposed as a solid core about which said collagen matrix is formed.
 9. The microsponge of claim 6 wherein said inert weighting material is selected from the group consisting of chromium, tungsten, cobalt, molybdenum, titanium, nickel and alloys thereof.
 10. The microsponge of claim 9 wherein said weighting material is titanium and said microsponge has hybridoma cells immobilized therein.
 11. A bioreactor system comprising a reactor vessel having aseptically sealed therein a plurality of sterilized weighted collagen microsponges for immobilizing bioactive materials in motive bioreactor systems, said microsponges comprising a porous, biostable, insoluble highly cross-linked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pore structure with an average pore size in the range of from about 1 to about 150 microns, the pores of said matrix occupying from about 70 to about 98% by volume of the microsponge, said microsponge also having an average particle size of from about 100 to about 1000 microns and a specific gravity of above about 1.05, wherein said highly crosslinked collagen matrix is prepared by the steps of(a) milling a source of collagen selected from the group consisting of Types I, II, and III collagen, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) freezing droplets of the weighted acid/collagen mixture to form a solid matrix, (e) vacuum drying said solid matrix to form a dry matrix; and (f) crosslinking the collagen in said dry matrix by a treatment selected from the group consisting of (i) contacting said collagen with a crosslinking agent selected from the group consisting of carbodiimides and bifunctional succinimidyl active esters, (ii) subjecting said collagen to elevated temperature under a vacuum, and (iii) a combination thereof.
 12. The biorector system of claim 11 wherein said reactor has a volume between about 0.1 to 10 liters.
 13. The bioreactor system of claim 12 wherein said reactor is a fluidized bed reactor, having a fluid distribution plate.
 14. A process for performing a bioreaction comprising immobilizing a bioactive material in weighted collagen microsponges comprising a porous, biostable, insoluble highly crosslinked collagen matrix containing an inert weighting material, said collagen matrix having an open to the surface pore structure with an average pore size in the range of from about 1 micron to about 150 microsn, the pores of said matrix occupying from about 70 to about 98% by volume of the microsponge, said microsponge also having an average particle size of from about 100 to about 1000 microns and a specific gravity above about 1.05; retaining the microsponges having said immobilized bioactive material in a suitable reactor vessel; passing a liquid reagent stream into said reactor in direct contact with said microsponges; agitating the mixture of said microsponges and said reagent stream and recovering the biochemical reaction products from said reactor, wherein said highly crosslinked collagen matrix is prepared by the steps of(a) milling a source of collagen selected from the group consisting of Tyypes I, II, and III collagen, (b) admixing said milled collagen with an acidic liquid medium, (c) adding inert weighting material to said acid/collagen mixture, (d) freezing droplets of the weighted acid/collagen mixture to form a solid matrix, (e) vacuum drying said solid matrix to form a dry matrix; and (f) crosslinking the collagen in said dry matrix by a treatment selected from the group consisting of (i) contacting said collagen with a crosslinking agent selected from the group consisting of carbodiimides and bifunctional succinimidyl active esters, (ii) subjecting said collagen to elevated temperature under a vacuum, and (iii) a combination thereof.
 15. The process of claim 14 wherein said bioactive material is organisms, the microsponges are incubated to promote growth and colonization of said microsponges by said organisms, said reagent comprises nutrient media for promoting the growth and metabolism of said organisms, and wherein said product comprises metabolites of said organisms.
 16. The process of claim 14 wherein said bioactive material is organisms and the recovered product comprises free organisms which have escaped from said microsponges.
 17. The process of claim 15 wherein said organisms comprise hybriodomas and said product comprises monoclonal antibodies.
 18. The process of claim 17 wherein said reactor vessel comprises a fluidized bed reactor.
 19. The process of claim 15 wherein said organisms comprise mammalian cells and said products comprise mammalian cell products.
 20. The process of claim 15 wherein said organisms are genetically engineered microbial cells and said product comprises secreted protein products.
 21. The process of claim 16 wherein said organisms are genetically engineered microbial cells and said product comprises said organisms containing a non-secreted protein product. 